The process of oncogenic transformation in vitro has been examined in an attempt to define the molecular mechanisms of carcinogenesis. Transformation in Ki-MSV-infected rat adrenal cortex cells appears to be a multistep process (Auersperg et al., 1981), as does the process of transformation in other nonestablished cells (Land et at., 1983b). The Ki-MSV-infected adrenal cortex cells initially express a partially transformed phenotype and after further passaging progress to a highly transformed phenotype (Calderwood and Auersperg, 1984). Examination of Ki-MSV-infected adrenal cortex cells indicated that progression to a highly transformed phenotype could occur in the absence of significant changes in the level of the expression of the viral ras oncogene. These results indicated that an oncogenically activated ras gene could be expressed in these nonestablished cells in the absence of transformation. Since ras and myc cooperate to transform primary fibroblasts the effect of the co-introduction of myc on Ki-MSV-induced transformation of adrenal cortex cells was examined. It could be demonstrated that myc and ras cooperate to transform the adrenal cortex cells more efficiently than either oncogene alone, but that the infected cultures initially only express some of the phenotypes associated with transformation. The appearance of a fully transformed phenotype, as monitored by growth in soft agar, was not expressed until several passages after infection. An analysis of the Ki-MSV/MMCV-infected cultures indicated that some of the phenotypes associated with activated oncogenes in immortalized cell lines appeared to be suppressed in the coinfected adrenal cortex cells. Transformation by ras and myc appears to require a further cellular change resulting in a loss of the suppression of oncogene action. The emergence of transformed cultures from the Ki-MSV-infected rat adrenal cortex cells was correlated with the reduced expression of a novel ras-related protein of 27000 Mr. Transformation induced by src and myc was also examined. These two oncogenes appeared to cooperate in a two step pathway of transformation that was not susceptible to cellular suppression. The transformed phenotype did not appear to be entirely free of external influence as the growth rate of the transformed cells could be modified by culture conditions. The ability of myc to cooperate with src and ras in the transformation of the early passage adrenal cortex cells provides further support for mutistep carcinogenesis. The effect of oncogenes on steroidogenesis was examined in the Y-1 adrenocortical tumour cell line. The effect of the virally borne oncogenes on growth and morphology of the Y-1 cells was relatively subtle. The oncogenes appear to stimulate the production of fluorogenic steroids, each in a distinct fashion. A model of transformation can be derived in which the roles of the oncogenes and their interaction with the cell can be evaluated. The differences in the pathways of transformation for the two combinations of oncogenes illustrates the potential complexity of the transformation process and provides an in vitro model system for further study. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Having the ability to perform large automatic software changes in a code base gives new possibilities for software restructuring and cost savings. The possibility of replacing software libraries in a semi-automatic way has been studied. String metrics are used to find equivalents between two libraries by looking at class- and method names. Rules based on the equivalents are then used to describe how to apply the transformation to the code base. Using the abstract syntax tree, locations for replacements are found and transformations are performed. After the transformations have been performed, an evaluation of the saved effort of doing the replacement automatically versus manually is made. It shows that a large part of the cost can be saved. An additional evaluation calculating the maintenance cost saved annually by changing libraries is also performed in order to prove the claim that an exchange can reduce the annual cost for the project.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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The bovine papillomaviruses (BPV) are capable of transforming cells from a wide range of species both in vivo and in vitro. The 69% portion of the BPV-1 viral genome that contains the transforming region, as well as the 31% portion, were cloned in the pBR322 plasmid vector. An extensive restriction endonuclease map of the transforming region was prepared. Using the cloned transforming region as a ³²P probe, BPV-1 coded mRNA transcripts from transformed cells were detected and sized using the northern blot technique. The largest open reading frame of the transforming region of the BPV-1 genome was sequenced using the Maxam and Gilbert chemical method and the amino acid sequence of a protein that this region could code for was presented.

The papillomaviruses do not appear to be capable of passage in cultured cell lines despite numerous attempts to identify conditions permissive for their propagation. These viruses induce benign tumors in animals (warts, papillomas) by unknown mechanisms. A broad range of animal species is susceptible to papillomavirus-induced tumorogenesis. The basic molecular mechanisms of papillomavirus replication, transcription, and translation to produce virus-specific products in cells are unknown. Since the viruses do not reproduce in vitro, no conditional-lethal and deletion mutants like those characterized in other systems exist. Thus, progress in understanding the papillomavirus-host cell interactions leading to neoplasia has been severely hindered. This dissertation is concerned with biochemical analysis of bovine papillomavirus type 1 (BPV-1) DNA and RNA in BPV-1 transformed and tumor cells. The specific conditions of infection resulting in stable lines of BPV-1 transformed cells are described. Colonies of BPV-1 transformed cells exhibiting anchorage-independent growth in agarose-containing medium were isolated and cloned cell lines were established. Formation of tumors in a rabbit following inoculation with BPV-1 is reported and represents the first evidence of BPV-induced tumorogenesis in this animal species. The BPV-1 transformed and tumor cells are characterized with respect to the quantity and physical state of the BPV-1 genome in the cells. The BPV-1 DNA is present in high copy numbers in free, non-integrated supercoiled and nicked open-circular forms. No evidence of integrated BPV-1 sequences is noted. This is unusual since all other characterized DNA tumor viruses require covalent integration of at least a portion of the viral DNA in the cellular genome during transformation. A detailed restriction endonuclease cleavage map of the BPV-1 genome is presented, representing a more complete physical characterization of the viral genome. The first evidence of BPV-1 specific transcripts in transformed cells is reported. These results should aid in functional characterizations of the BPV-1 genome, particularly the determination of the specific regions of the BPV-1 DNA transcribed during transformation of cells, analogous to early regions defined in other DNA tumor virus systems, and analysis of post-transcriptional processing mechanisms involved in the synthesis of BPV-1 specific messenger RNA.

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